The present invention is concerned with compositions and methods that rely upon the tetracycline resistance (tet) operator and repressor to control transcription in mammalian cells. It encompasses methods for recombinantly producing proteins and the vectors and host cells utilized in such methods. In addition, the present invention is directed to viruses which are recombinantly engineered so that their replication is controlled by the tet operator/repressor system. These viruses may serve as vehicles for gene transfer both in vitro and in vivo; as agents for immunization; and as a means for delivering nucleic acid therapeutic agents to cells.
The ability to specifically regulate transgene expression has been a central concern in molecular biology for many years. In the case of mammalian cells, the in vitro regulation of recombinant genes has most often been accomplished through the use of inducible promoters that respond to agents such as heavy metal ions (Brinster, et al., Nature 296:3942 (1982); heat shock (Nover, in Heat Shock Response, pp. 167-220, CRC, Fla. (1991)); and hormones (Klock, et al., Nature 329:734-736 (1987)). Unfortunately, these promoters generally provide only a relatively a low level of expression even in the presence of inducer and most of the inducers that have been used in vitro.have unacceptable side effects in vivo.
As an alternative to inducible promoters, attempts have been made to control mammalian gene expression using well-characterized prokaryotic regulatory elements. In most cases, regulatory systems have relied upon strong interactions between prokaryotic operators and repressor proteins as a means for either targeting eukaryotic transcription modulators to specific sites within a host cell genome (see e.g., Labow, et al., Mol. Cell. Biol. 10:3343-3356 (1990)) or in attempts to directly inhibit gene expression using the prokaryotic repressor (see e.g., Brown, et al., Cell 49:603-612 (1987)).
In the case of prokaryotic elements associated with the tetracycline resistance (tet) operon, systems have been developed in which the tet repressor protein is fused with polypeptides known to modulate transcription in mammalian cells. The fusion protein has then been directed to specific sites by the positioning of the tet operator sequence. For example, the tet repressor has been fused to a transactivator (VP16) and targeted to a tet operator sequence positioned upstream from the promoter of a selected gene (Gussen, et al., Proc. Nat""l Acad. Sci. USA 89:5547-5551 (1992); Kim, et al., J. Virol. 69:2565-2573 (1995); Hennighausen, et al., J. Cell. Biochem. 59:463472 (1995)). The tet repressor portion of the fusion protein binds to the operator thereby targeting the VP16 activator to the specific site where the induction of transcription is desired. An alternative approach has been to fuse the tet repressor to the KRAB repressor domain and target this protein to an operator placed several hundred base pairs upstream of a gene. Using this system, it has been found that the chimeric protein, but not the tet repressor alone, is capable of producing a 10 to 15-fold suppression of CMV-regulated gene expression (Deuschle, et al., Mol. Cell. Biol. 15:1907-1914 (1995)). The main problem with these types of systems is that the portion of fusion proteins corresponding to the mammalian transactivator or repressor tends to interact with cellular transcriptional factors and cause pleiotropic effects.
Ideally, a system for regulating mammalian gene expression should be highly specific for a selected gene and subject to induction by factors suitable for use both in vitro and in vivo. The present invention discloses such a system and describes how it can be used to regulate transgene expression. In addition, the invention describes how this system can be adapted to engineer viruses to serve as vectors, therapeutic agents and vaccines.
The present invention is directed to a number of different compositions and methods which share the common feature of having gene expression regulated by the tet operator/repressor system.
A. Compositions and Methods for the Production of Recombinant Protein
In its first aspect, the invention is directed to a recombinant DNA molecule which contains a mammalian promoter sequence with a TATA element; at least one tet operator sequence; and a gene sequence operably linked to the promoter and lying downstream from the operator. The exact positioning of the operator sequence (or sequences) relative to the TATA element is critical to the invention. In order to be effective at controlling transcription, the operator must begin at least 6 nucleotides downstream from the last nucleotide in the TATA element and, when a gene encoding a protein is expressed, the operator should be positioned before the translation initiation codon. In general, the operator should not begin more than about 100 nucleotides downstream and, preferably, it should begin within 6 to 24 nucleotides downstream of the TATA element. When positioned in this manner, it has been found that the binding of the repressor protein causes an essentially complete shutdown in transcriptional activity. This is true even for very strong and highly promiscuous promoters such as the human CMV immediate early promoter.
It is expected that the recombinant DNA molecule described above will, most typically, be incorporated into mammalian cells that constitutively express the tet repressor protein. Suitable cells may be developed by transforming a mammalian cell line, e.g., U2OS cells or Vero cells, with a vector containing the tet repressor protein gene operably linked to a promoter active in the cells (e.g., a CMV promoter, HSV-1 promoter or SV40 promoter). Alternatively, the DNA molecule may contain, in addition to the elements already discussed, a second promoter, preferably constitutive, operably linked to the tet repressor gene sequence. The invention encompasses, not only the DNA molecules, but also the host cells transformed with the DNAs and the recombinant proteins made by the cells.
The present invention is also directed to a method for recombinantly producing protein in which mammalian host cells are transformed with a vector containing a mammalian promoter sequence having a TATA element; at least one tet operator sequence positioned at least 6 nucleotides 3xe2x80x2 to the TATA element; and a gene lying 3xe2x80x2 to the operator and operably linked to the promoter. The gene 3xe2x80x2 to the operator may encode an antisense nucleic acid that inhibits the expression of a selected gene, a therapeutically active agent (e.g. a tumor suppressor or a transdominant negative mutant polypeptide of a cellular protein), a protein of interest for experimental purposes or simply a protein whose isolation is desired. In all cases where the gene encodes a protein, the operator sequence will be positioned before the translation initiation codon of the gene. The transformed cells should constitutively express the repressor protein and recombinant gene expression may be induced in the cells by introducing tetracycline. Typically, the tet operator sequence will be located between 6 and 100 nucleotides (preferably between 6 and 24 nucleotides) 3xe2x80x2 to the last nucleotide in the TATA element. The preferred promoter is the human CMV immediate-early promoter. It has been found that this system allows for the very tight regulation of gene expression, i.e., expression is essentially completely shut off until the inducer, tetracycline, becomes available.
The method can be used to produce recombinant protein in cultured mammalian cells or in the cells of a transgenic or non-transgenic animal. When a transgenic animal is used for production, it will most typically be a mouse and it is necessary that the cells transformed with the vector described above be embryonic stem cells. The stem cells may be engineered to express the tetracycline repressor by transforming them with the repressor gene operably linked to a promoter prior to transformation with the tet operator and recombinant gene. Alternatively, the repressor gene can be incorporated into the same DNA construct as the tet operator and placed under the control of either the same promoter as the gene encoding the recombinant protein or under the control of a separate promoter. The transformed stem cells are incorporated into a blastocyst to form a chimeric embryo, which is implanted into a pseudopregnant animal. Embryos implanted in this manner are allowed to develop into viable offspring that are screened to identify heterozygous animals expressing the recombinant gene. The heterozygous animals are then bred to produce homozygous animals that make recombinant protein in response to the administration of tetracycline.
The invention encompasses the transgenic animals made using this method and any transgenic animal that has integrated into its genome recombinant DNA containing a mammalian promoter sequence having a TATA element; at least one tet operator sequence positioned at least 6 nucleotides 3xe2x80x2 to the TATA element; and a gene lying 3xe2x80x2 to the operator and operably linked to the promoter. When the gene encodes a protein, the sequence of the operator will be positioned before the translation initiation codon of the gene. Typically, the tet operator sequence will be located between 6 and 100 nucleotides (preferably between 6 and 24 nucleotides) 3xe2x80x2 to the last nucleotide in the TATA element. The preferred promoter is the human CMV immediate-early promoter. In addition to the transgenic animals, the invention encompasses the recombinant proteins made by these animals.
B. Engineered Viruses and Their Uses
One particularly important use of the tet operator/repressor expression system is in the making of viruses in which replication can be controlled. The essential characteristic of these viruses is that they contain within their genome at least three related elements: a recombinant promoter having a TATA element; at least one tet operator sequence positioned at least 6 nucleotides 3xe2x80x2 to the TATA element; and a gene operably linked to the promoter, which lies downstream from the operator and which inhibits viral replication when expressed. Typically, the tet operator sequence will be located between 6 and 100 nucleotides (preferably between 6 and 24 nucleotides) 3xe2x80x2 to the last nucleotide in the TATA element. The gene lying downstream of the operator may act either by encoding a protein that inhibits viral replication or by forming a transcription product that inhibits viral replication through an antisense mechanism. When the gene encodes a protein, the tet operator sequence will be positioned upstream from the translation initiation codon. The engineered virus can be made and grown in cultured cells that constitutively express the tet repressor protein. Under these conditions, the gene that inhibits viral replication will be shut off, allowing large amounts of virus to be produced. Virus may then be collected, purified, and introduced into mammalian cells either in vitro or in vivo. Since mammalian cells do not normally make the tet repressor protein, the operator sequence will be unoccupied. As a result, the gene lying 3xe2x80x2 to the tet operator is expressed and viral replication is prevented.
Viruses engineered in the manner discussed above have a wide range of possible applications. First, the viruses can be used as a vehicle for delivering DNA, (e.g., a gene) to mammalian cells. Under these circumstances, a second recombinant promoter will typically be incorporated into the viral genome and operably linked to the gene whose expression is desired. This second promoter may or may not, be followed by one or more tet operators lying between 6 and 100 (preferably between 6 and 24) nucleotides downstream from a TATA element in the second recombinant promoter. After having delivered the DNA to the host cell, production of new virus is inhibited due to the absence of the tet repressor protein. The gene attached to the second promoter may encode an antisense nucleic acid that inhibits the expression of a selected gene within cells; a therapeutically active protein (e.g., a tumor suppressor or a transdominant negative mutant polypeptide of a cellular protein); or simply a protein that will be isolated or that is of interest for experimental reasons. The invention encompasses the method of transforming host cells by transfecting them with the virus, the transformed host cells themselves and the recombinant proteins made by the host cells.
The viruses discussed above may also be used to immunize subjects. The great advantage of vaccines containing the engineered viruses that, because the viruses will not replicate after they are injected into subjects, the risk of active viral infection due to immunization is greatly reduced. To further ensure that virus replication will not occur, additional mutations may be introduced into the viruses, e.g. a deletion mutation may be introduced into one or more essential viral genes. In general, viruses containing such additional mutations will be preferred.
The engineered viruses also have utility in the direct treatment of patients for viral infections. The first step in this method involves transforming a second virus (i.e., a virus other than the one that has infected the patient although possibly of the same strain) by incorporating into its genome: DNA comprising a mammalian promoter with a TATA element; at least one tet operator sequence positioned at least 6 nucleotides 3xe2x80x2 to the TATA element (typically between 6 and 100 nucleotides 3xe2x80x2 to the TATA element); and a gene positioned 3xe2x80x2 to the operator and operably linked to the promoter. This gene should be chosen so that, when expressed, it is capable of blocking the replication of both the second virus and the virus which has infected the patient. In cases where the gene encodes a protein, the sequence of the tet operator will be positioned before the translation initiation codon of the gene. The transformed second virus is grown in host cells expressing the tet repressor protein, thereby allowing large amounts of viral progeny to be produced. Virus is collected, purified and then administered to the patient. In preferred embodiments, the tet operator is located between 6 and 24 nucleotides downstream from the last nucleotide in the TATA box and the promoter used is the human CMV immediate-early promoter.
Finally, the present invention is directed to a method for delivering a nucleic acid therapeutic agent to cells. The nucleic acid therapeutic agent may comprise either an antisense fragment that inhibits the expression of a cellular protein, or a gene that encodes a protein with a therapeutic action. The virus is engineered to contain within its genome: i) a recombinant mammalian promoter with a TATA element; at least one tet operator sequence positioned at least 6 nucleotides 3xe2x80x2 to the TATA element and 5xe2x80x2 to a translation initiation codon; and iii) a gene positioned 3xe2x80x2 to the operator and operably linked to the promoter. When this gene is expressed, viral replication is inhibited. Typically, the tet operator sequence will be located between 6 and 100 nucleotides (preferably between 6 and 24 nucleotides) 3xe2x80x2 to the last nucleotide in the TATA element. The preferred promoter is the immediate-early promoter of human CMV. In addition, the virus must contain within its genome the nucleic acid encoding the therapeutic agent operably linked to a second promoter. This second promoter may, or may not, be followed by one or more tet operators lying, typically, between 6 and 100 (preferably between 6 and 24) nucleotides downstream from a TATA element in the second promoter.
After the preparation of the viral vector for delivering therapeutic agent, the next step in the method is to grow a large amount of the virus in a host cell that expresses the tet repressor protein. The virus grown in this manner is collected, purified and then administered to the patient. Since the patient would not normally have cells synthesizing tet repressor, replication of virus will be blocked but transcription of the nucleic acid therapeutic agent will proceed.